Filtration

ABSTRACT

Filtration methods comprise virus-filtering a solution containing a least one macromolecule. The total salt content of the solution for virus-filtering is within the range of from about 0.2 M up to saturation with the salt. Salts that can be used in the filtering methods include sodium chloride, potassium chloride, sodium acetate, sodium citrate, sodium phosphate, potassium dyhydrophosphate and combinations thereof.

TECHNICAL FIELD

The present invention relates to a method of virus-filtering a solutionthat contains at least one macromolecule, by virtue of the total saltcontent of the solution lying in the range of from about 0.2 M up tosaturation of the solution with the salt concerned. The inventive methodreduces the residence time and the extent to which the solution need tobe diluted, and optimizes the yield when virus-filtering primarilyproteins, polysaccharides and polypeptides. The reduction in viruscontent is at least as good as with conventional techniques where thetotal salt content is low. The present invention facilitates virusfiltration with the aid of the so-called “dead-end” technique, whichaffords several process and economic advantages in comparison with thetangential virus-filtering technique normally used. When virus-filteringthe plasma protein factor IX, the yield obtained in the virus-filteringstage is increased from about 70% to above 95%, by raising the saltcontent of the solution in accordance with the present invention.

BACKGROUND OF THE INVENTION

The problem of virus contamination of various protein preparationsintended for the medication of human beings has received greater noticein recent years. For instance, occasional reports have been submittedconcerning, e.g., blood proteins that have been contaminated withhepatitis virus A, hepatitis virus B, hepatitis virus C and/or HumanImmunodeficiency Virus (HIV). In keeping with these reports, theauthorities of several countries have sharpened their requirements withregard to cleansing protein preparations of their possible viruscontaminants.

In present-day, conventional techniques, viruses are inactivated withthe aid of chemical additives, primarily solvents and detergents, and/orby exposing the viruses to elevated temperatures. The former method hasthe drawback of functioning solely on virus with lipid envelopes, forinstance hepatitis virus B and HIV. The latter technique mentioned abovehas the drawback that many proteins are thermally unstable at thosetemperatures required to effectively reduce the contaminating virus.

U.S. Pat. No. 4,473,494 (assigned to the U.S. Secretary of the Army)discloses a method for production of stroma-free, non-heme protein-freehemoglobin by use of zinc ions to promote precipitation of a zincion-bound insoluble hemoglobin complex, followed by membraneultrafiltration of the zinc-hemoglobin complex from the filtrate fluidmedium. In the only step where viruses are said to be removed fromhemoglobin, the total salt content is below 0.05 M, i.e. the totalcontent of salt is conventional.

EP-A-0307373 (assigned to Ares-Serono) relates to removal of virusesand/or other contaminants from biological materials in fluid form byusing ultrafiltration membranes having a 100,000 Da cut-off. A preferredbiological material is human growth hormone. In the examples ofEP-A-0307373, the total content of salt in the virus-filtering step liesin the range of from 0.01 up to 0.10 M (NH₄CO₃), i.e. the total contentof salt is conventional.

There is thus a need for an effective virus-reducing method which can beapplied to different types of macromolecules, primarily proteins, and ondifferent types of viruses.

DESCRIPTION OF THE INVENTION

One object of the present invention is to markedly reduce the residencetime when virus-filtering solutions that contain macromolecules.

Another object of the present invention is to markedly reduce the liquidvolumes when virus-filtering solutions that contain macromolecules.

A further object of the present invention is to reduce the filter arearequired to effectively virus-filter solutions that containmacromolecules.

Yet another object of the present invention is to achieve amacromolecule yield in excess of about 90% in the virus-filtering stage.

Still another object of the present invention is to reduce thepolymerization obtained on the virus filter surface, so as to enable therate of flow to be increased and the process time to be decreased.

These and other objects are fulfilled by the present invention, whichrelates to a method of virus-filtering a solution containing at leastone macromolecule wherein the total salt content of the solution lieswithin the range of from about 0.2 M up to saturation of the solutionwith the salt concerned.

The inventor of this invention has thus found that virus filtration canbe effected much more effectively than previously known, by increasingthe salt content of the solution. This discovery is surprising, becausehitherto in virus filtration of proteins it has been believed thatsolely the protein concentration, the rate of flow and the pH have hadany influence on the process.

It is believed that the enhanced filtering effect achieved at highersalt concentrations is because the protein contracts and can therewithpass more easily through the filter pores. It is also conceivable, thatthe interaction is reduced between macromolecules themselves and/orbetween the macromolecules and the material of the filter membrane. Itis also conceivable that proteins having a large number of hydrophobicgroups are influenced to a greater extent by an elevated saltconcentration.

The closer the molecular weight, or relative molecular mass, of themacromolecule lies to the pore size of the filter membrane, the moreeffective the present invention. The effectiveness of the presentinvention is also enhanced when the difference in the size and/or themolecular weight of the contaminants and the product increases, i.e.with increasing concentrations of high molecular contaminants in theproduct.

The present invention also facilitates specific fractions to beseparated from a desired product, for instance enables undesirableproteins to be separated from the protein that constitutes the product.

The use of a high salt content according to the present invention, alsoenables the use of the so-called “dead-end” filtering technique. Thispreferred embodiment, has several advantages over conventionaltangential filtering processes normally applied, especially with a poresize of about 5-30 nm. For instance, the equipment and operatingprocedures required are much simpler and therewith less expensive. Theuse of “dead-end” filtration also reduces the loss of the macromolecule,reduces the process time, increases the permeability of themacromolecule through the filter, and also enables a generally constantconcentration of the macromolecule to be achieved over the filter aswell as a constant membrane pressure. Another advantage with thedead-end filtering technique, is the fact that scaling-up of virusfiltering processes from laboratory to industrial scale is considerablyfacilitated.

When practicing the present invention, the total salt content of thesolution suitably lies within the range of from 0.3 up to 3.0 M,preferably within the range of from 0.4 up to 2.5 M, and more preferablywithin the range of from 0.6 up to 2.0 M. It is particularly preferredthat the total salt content of the solution lies within the range offrom 0.8 up to 1.5 M.

When necessary, the total salt content of the solution can be adjustedby adding any acceptable salt. For instance, it is possible to usesoluble inorganic salts, soluble organic salts or combinations of suchsalts. It is assumed that important process advantages are obtained whenusing salts which exhibit a high salting-out effect in accordance withthe so-called Hofmeister series. Reference is here made to S. Glasstone,Textbook of Physical Chemistry, van Nostrand Co., Toronto, 2^(nd)edition, April 1946, pp. 1254-1259. The most important examples ofanions which have such high salting-out effect are citrate, tartrate,sulfate, acetate and phosphate. Cations that can be used advantageouslywhen practicing the present invention are monovalent cations, such assodium, potassium and ammonium, as well as divalent cations, such ascalcium. Sodium chloride, potassium chloride, sodium acetate and sodiumcitrate or combinations thereof are particularly preferred salts inaccordance with the invention, in view of the advantages that areafforded by pharmaceutically acceptable additives. It is alsoconceivable to add one or more salts in sequence, when the filtrationprocess is carried out in two or more steps.

A protein concentration within the range of from about 5 up to about 10mg/ml solution is often recommended for virus filtration. When applyingthe present invention, it was surprisingly found that solutions having ahigher protein concentration, from about 10 up to about 20 mg/ml, couldbe processed advantageously through the virus filter.

The solution should have a temperature within the range from 0° C. up tothe temperature at which the protein concerned is denatured. Thetemperature of the solution suitably lies within the range of from 10°C. up to 50° C., preferably from 20° C. up to 35° C.

When practicing the present invention, the solution should have a pH inthe range of from about 3 up to about 9, suitably from 4 up to 8. The pHof the protein solution should not lie too close to the isoelectricpoint of the protein concerned. For instance, in the case ofgammaglobulin, a better result is obtained with a pH of 5.5 than with apH of 6.8.

In the present invention, solution refers to a solution that contains atleast 50 percent by weight of water, optionally including one or moresolvents, such as methanol, ethanol, acetone or acetonitrile.

The present invention can be used to optimize process procedures whenvirus-filtering solutions that contain a large number of different typesof macromolecules. Examples of such molecules are proteins,polysaccharides and polypeptides or combinations thereof. The origin ofthe macromolecules is irrelevant to the use of the present invention.The macromolecules may thus derive from the plant kingdom or the animalkingdom or may be produced initially by industrial processes. However,the macromolecules are suitably of human or animal origin or engineeredgenetically (recombinants).

Particularly appropriate proteins in regard of the present invention arefactor VIII, factor IX, antithrombin III, gammaglobulin, albumin,streptokinase, apolipoproteins and growth hormones.

A particularly preferred factor IX product is Nanotiv®, which issupplied by Pharmacia AB, Stockholm, Sweden. The advantage with thisproduct is that its specific activity prior to filtration issufficiently high to enable the use of a filter of very fine structure.This enables the virus concentration to be lowered to an extremely lowlevel, at the same time as the filtering process itself is very rapidand produces a high yield.

Preferred types of factor VIII are deletion derivatives of recombinantproduced factor VIII products. A particularly preferred factor VIIIproduct is r-VIII SQ supplied by Pharmacia AB, Stockholm, Sweden. Oneadvantage with this product is that the recombinant produced productmolecule lacks the inactive intermediate part of the natural factor VIIImolecule. This gives the molecule a mean molecular weight of about170,000. A molecule of this size is particularly suited for filtrationwith such filters as those which enable a considerable virus reductionto be achieved.

Preferred apolipoproteins include apolipoprotein AI (Apo AI),apolipoprotein AII (Apo AII), apolipoprotein AIV (Apo AIV),apolipoprotein E (Apo E) and variants or mixtures thereof. Variantsinclude preforms, fragments and truncated, extended or mutated forms ofApo AI, Apo II, Apo IV and Apo E. Mutated forms in which at least onearginine group has been replaced with a cystein group are particularlypreferred. One such mutated form is Apo A-IMilano (Apo A-IM), alsoproduced with recombinant DNA technique by Pharmacia AB, Stockholm,Sweden.

Polysaccharides which are particularly preferred in accordance with thepresent invention are glycosaminoglycans and bacteria polysaccharides.Examples of glycosaminoglycans are heparins, heparin fragments, heparinderivatives, heparan sulfate and hyaluronic acid. A particularlypreferred group of glycosaminoglycans is comprised of low molecularweight heparins having a mean molecular weight of up to about 10,000,preferably from 2,000 up to 8,000.

According to the present invention, particularly suitable polypeptidesare bioactive polypeptides, such as recombinant human growth hormonesproduced in mammalian cells.

The present invention can thus be used to optimize the process ofvirus-filtering solutions that contain, e.g., proteins, polysaccharidesand polypeptides. However, the invention is described in the followingwith reference to solutions that contain proteins, more specificallyproteins that occur naturally in the human organism.

Those viruses that may be present in protein solutions will normally bemuch larger than the proteins themselves. It is thus presumable thatviruses can be removed from proteins in accordance with size, forinstance by filtration.

Viruses that can be removed efficiently with the present invention, canhave a size smaller than about 350 nm. The size of the viruses that canbe removed, suitably is smaller than 200 nm, preferably smaller than 150nm. Normally, the viruses that can be removed are larger than about 20nm, i.e. the approximate size of the parvo virus.

The present invention is primarily intended for removing viruses frommacromolecules, where the macromolecules are the product of interest. Itis, however, within the scope of the invention, to use the presentmethod for separating viruses from macromolecules, where the viruses arethe product of interest. An example, is the purification of parvovirusfor use as a testing agent, and poliovirus for use a vaccine, whereine.g. proteins and polysaccharides can be removed by the present method.

Virus filtration is normally carried out in a tangential filteringprocess or in a so-called “dead-end” filtering process. In tangentialvirus filtration, the protein solution is pumped around at a constantrate of flow on the retention side, while another pump draws the proteinsolution through the filter by suction. When a given volume has beenobtained on the retention side, a buffer is added on the retention side.This procedure is repeated a number of times, as necessary, with themajor part of the remaining protein passing through the filter whileretaining the virus on the retention side. Such a process is calleddiafiltration. The filter is normally discarded after each run, to avoidtransferring the virus.

In the case of so-called “dead-end” virus filtration, the same virusfilter as that used in tangential virus filtration can be used, althoughthe peripheral equipment and operating procedures are much simpler andless expensive than in the case of tangential virus filtration. Thus, inprinciple, “dead-end” filtration involves placing themacromolecule-containing solution in a pressure vessel prior tofiltration and pressing the solution through the virus filter with theaid of a pressure source, suitably nitrogen (gas).

The degree of fineness of filters generally, is normally given as poresize or the approximate molecular weight (relative molecular mass) atwhich the molecules are stopped by the filter, the so called cut-off. Inthe present invention, the virus filters can have a cut-off of about1,000,000, suitably 500,000. To remove small viruses, the virus filtersshould have a cut-off of 200,000, preferably 100,000. To reach a maximumvirus-reduction, the virus filter should have a cut-off slightly higherthan the macromolecule which is virus-filtered.

Virus filters are known in the art and are supplied by Millipore fromMassachusetts, USA and Asahi Chemical Industry Co., Ltd. from Japan,among others. Millipore supplies filters having two different types ofmembrane, depending on the size of the protein concerned. For instance,Millipore supplies, among others, Viresolve™/70 for proteins having amolecular weight, or relative molecular mass, of up to about 70,000, andViresolve™/180 for proteins having a molecular weight of up to about180,000. This latter filter can be used for monoclonal antibodies, forinstance. Asahi Chemical Industry supplies, among other things, Planova™35 and Planova™ 15 filters, this latter filter being used to removesmaller viruses, such as the polio virus.

As mentioned before, the choice of filter will depend on the size of theprotein concerned, among other things. Factor IX, antithrombin III,human serum albumin (HSA) and Apo A-IM (the dimer) all have a molecularweight of roughly 60,000-70,000, wherein Viresolve™/70, for instance, isa suitable alternative. Gammaglobulin has a molecular weight of about180,000, wherein Viresolve™/180, for instance, is a suitablealternative. The latter filter is also suitable for use with therecombinant produced factor VIII product, r-VIII SQ, which has amolecular weight of about 170,000, as mentioned before.

The possibility of choosing a fine structure filter also assumes thatthe protein solution has a high degree of purity prior to filtration. Inturn, the use of a fine structure filter is a prerequisite for theability to produce protein solutions which have a very low virus contentin the end product. Thus, in order to be able to reduce the virusconcentration to a very low level, there is required a filter of veryfine structure, for instance Viresolve™/70. The virus concentrationcannot be lowered to quite such a low level when using Viresolve™/180.

The effectiveness, or efficiency, of the filtering stage is influencedby the purity of the protein solution delivered to the filter. In thisregard, a high specific activity prior to filtration results in a higheryield in the filtering stage. For instance, in the case of preferredembodiments applied when filtering solutions that contain factor IX, ithas been found that the protein yield in the filtering stage can beincreased from about 70% to above 95%. However, when practicing thepresent invention, it is possible to achieve protein yields of above90%, even when working with solutions of low specific activity.

With the present invention, it is possible to reduce the content of verysmall non-enveloped viruses, such as the parvovirus, by at least 3 logs,suitably at least 4 logs, and preferably at least 5 logs. The reductionis very good with the tangential technique, but even better with the“dead-end” technique, when applied according to the present invention.

According to the invention, virus filtration is preferably carried outat the end of a protein manufacturing sequence, since a high specificactivity prior to filtration will result in a higher protein yield inthe filtering stage. The present invention is preferably applied as alast purification stage, optionally followed by a stage for adjusting,for instance, protein concentration, salt content or the pH of the endproduct. A following diafiltration stage using a UF-membrane may also beapplied to remove salts which although advantageous from a process oreconomic aspect during virus filtration should not be included in theend product. Protein solutions which are ready for administration willnormally contain a physiological solution, for instance 0.15 M sodiumchloride at a pH of 7, in combination with one or more stabilizers, suchas saccharose or amino acids. The virus filtration process may also becarried out in two or more steps, with or without intermediate processsteps.

The present invention effectively reduces the content of virus withlipid envelopes and viruses without lipid envelopes. Examples of viruseswithout a lipid envelope are the hepatitis virus A, polio virus andparvo virus, which, are relatively small viruses. Examples of viruseswith a lipid envelope are the hepatitis virus B, the hepatitis virus Cand the Human Immunodeficiency Virus (HIV).

The invention will now be illustrated in more detail with the aid ofexemplifying, non-limiting examples.

Experimental Section

Experiments were carried out in which the sieving coefficient ofproteins, or protein permeability factor, was first determined atdifferent filtrate flowrates. The sieving coefficient, or proteinpermeability factor, is given as P/R, where P is the concentration ofprotein on the permeate side (the filtrate side) measured by absorptionat 280 nm (A₂₈₀) and R is the concentration of protein on the retentionside (R) measured by absorption at 280 nm (A₂₈₀). The filtrate flowratewhich gave the highest sieving coefficient in the absence ofpolymerization on the filter was then chosen. A yield optimization wasalso made with some macromolecules.

EXAMPLE 1

Experiments were carried out with factor IX as the macromolecule, toillustrate the effect of two salt contents on the protein sievingcharacteristic, the diafiltration volume and the yield. A commercialsolution containing factor IX, Nanotiv®, was supplied by Pharmacia AB,Stockholm, Sweden. The solution containing factor IX was obtained fromhuman blood plasma and prior to filtration had been treated in asequence involving anion exchange, chemical virus inactivation, affinitychromatography and cation exchange. The solution was ultra-filteredbetween each stage, except between the chemical virus inactivating stageand the affinity chromatographic stage.

Experimental Conditions:

-   Degree of purity of the entering protein solution: high.-   Buffer: 0.144 M NaCl+0.0055 M sodium citrate.-   Total salt content: about 0.15 M.-   Protein concentration: 0.5-1.0 A₂₈₀ units.-   Protein solution pH: 7.-   Experimental temperature: room temperature (about 23° C.).-   Virus separating filters: Viresolve™/70.-   Filtering technique: tangential.-   Filter area: ⅓ ft²-   Retention flowrate: 41 l/h.-   Pump: Watson-Marlow 504.

Transmembrane pressure: 0.2-0.3 bar. TABLE 1 Determining the proteinsieving coefficient. Experiment Filtrate flowrate, ml/min P/R, % 1 3.535.0 2 6.9 39.6 3 10.7 45.8 4 14.1 56.2 5 17.6 55.6 6 20.8 58.3 7 24.361.7

An optimal filtrate flowrate of 20.8 ml/min was obtained by determiningthe protein sieving coefficient. TABLE 2 Yield optimization. Filtrateflowrate: 20.8 ml/min. High degree of protein solution purity. Buffer:0.144 M NaCl + 0.0055 M sodium citrate. Total salt content: about 0.15M. Experiment Filtration time P/R, % 1  3 min 10 s 55.1 2  6 min 25 s52.1 3 10 min 40 s 44.5 4 13 min 20 s 34.0

Diafiltration with a dilution of about 1 volume unit per volume unit ofentering protein solution (1+1) resulted in a yield of about 90%.

EXAMPLE 2

The same conditions were applied as those applied in Example 1, with theexception that in this case the buffer comprised 1.0 M NaCl+0.01 Msodium citrate, which gave a total salt content of about 1.0 M. TABLE 3Determining the protein sieving coefficient. Experiment Filtrateflowrate, ml/min P/R, % 1 3.5 55.2 2 6.9 55.7 3 10.7 61.4 4 14.1 68.4 517.6 74.2 6 20.8 77.0 7 24.3 80.5

This determination of the protein sieving coefficient gave an optimalfiltrate flowrate of 24.3 ml/min. TABLE 4 Yield optimization. Filtrateflowrate: 24.3 ml/min. Experiment Filtration tim P/R, % 1 2 min 30 s 722 — 68 3 7 min 14 s 65 4 9 min 38 s 55

Diafiltration with a dilution of about 0.3 volume units per volume unitof entering solution (1+0.3) resulted in a yield of >95%.

EXAMPLE 3

The virus removing effect achieved with the experiments disclosed inExamples 1 and 2 was determined by a virus study. The study was carriedout on parvovirus, which are non-lipid-enveloped viruses and which havea size of 20-25 nm. In principle, experiments with such viruses fallinto the “worst case” category since they are some of the smallestviruses known.

The parvovirus was added to the solutions containing factor IX, with asalt content of 0.144 M NaCl+0.0055 M sodium citrate (experiment 1) and1.0 M NaCl+0.01 M sodium citrate (experiment 2) respectively. Thesolutions were then virus-filtered in accordance with Examples 1 and 2.The solutions were analyzed with respect to the parvovirus both beforeand after virus filtration. Experiment Virus reduction 1 1 × 10^(3.7) 21 × 10^(4.0)

The results show that virus filtration in accordance with Examples 1 and2 fulfil the requirements placed by the authorities on the virusreduction in one process step. Furthermore, the use of a high saltcontent in accordance with the invention is at least equally aseffective in removing virus as previously known techniques.

EXAMPLE 4

The same conditions were applied as those applied in Example 1, with theexception that the entering protein solution was not as pure.

Diafiltration with dilution of about 3 volume units per volume unit ofentering protein solution (1+3) resulted in a yield of about 65%.

EXAMPLE 5

The same conditions were applied as those applied in Example 2, with theexception that the entering protein solution was not as pure.

Diafiltration with dilution of about 3 volume units per volume unit ofentering protein solution (1+3) resulted in a yield of 89%. The yield offactor IX:C was 87%.

EXAMPLE 6

Experiments were carried out with factor IX as the macromolecule, toshow the effect of four salt contents on the protein sievingcoefficient, the diafiltration volume and the yield, with otherexperiment conditions being constant. The Nanotiv® solution used wassimilar to that used in Example 1. The experimental conditions appliedwere the same as those applied in Example 1. TABLE 5 Determining theprotein sieving coefficient. The buffer comprised 0.144 M NaCl + 0.0055M sodium citrate. Total salt content: about 0.15 M. Experiment Filtrateflowrate, ml/min P/R, % 1 3.5 25 2 6.9 28 3 14.1 43 4 20.8 49 5 24.3 50

TABLE 6 Determining the protein sieving coefficient. The buffercomprised 0.5 M NaCl + 0.01 M sodium citrate. Total salt content: about0.5 M. Experiment Filtrate flowrate, ml/min P/R, % 1 3.5 36 2 6.9 44 314.1 61 4 20.8 67 5 24.3 69

TABLE 7 Determining the protein sieving coefficient. The buffercomprised 1.0 M NaCl + 0.01 M sodium citrate. Total salt content: about1.0 M. Experiment Filtrate flowrate, ml/min P/R, % 1 3.5 49 2 6.9 60 314.1 72 4 20.8 74 5 24.3 76

TABLE 8 Determining the protein sieving coefficient. The buffercomprised 1.5 M NaCl + 0.01 M sodium citrate. Total salt content: about1.5 M. Experiment Filtrate flowrate, ml/min P/R, % 1 3.5 48 2 6.9 56 314.1 73 4 20.8 76 5 24.3 74

It is evident from Tables 5 to 8 that the present invention provides amarked improvement in the process conditions when virus-filtering factorIX solutions in comparison with previously known techniques where lowsalt contents have been used.

EXAMPLE 7

Experiments were carried out with factor IX as the macromolecule to showthe effect of three different salts on the protein sieving coefficient,the diafiltration volume and the yield, with other experiment conditionsbeing constant. The Nanotiv® solution used was similar to that used inExample 1. The conditions applied were the same as those applied inExample 1. TABLE 9 Determining the protein sieving coefficient. Thebuffer comprised 0.5 M potassium dihydrophosphate. Total salt content:0.5 M. Experiment Filtrate flowrate, ml/min P/R, % 1 3.5 34 2 6.9 48 314.1 57 4 20.8 55

TABLE 10 Determining the protein sieving coefficient. The buffercomprised 0.5 M NaCl. Total salt content: 0.5 M. Experiment Filtrateflowrate, ml/min P/R, % 1 3.5 27 2 6.9 43 3 14.1 50 4 20.8 46

TABLE 11 Determining the protein sieving coefficient. The buffercomprised 0.5 M barium chloride. Total salt content: 0.5 M. ExperimentFiltrate flowrate, ml/min P/R, % 1 3.5 24 2 6.9 36 3 14.1 34 4 20.8 —

It will be evident from Tables 9 to 11 that the present invention can becarried out advantageously with a number of different salts. It willalso be seen that the protein sieving coefficient increases when usingsalts that have a high salting-out effect in accordance with theHofmeister series (potassium dihydrophosphate) in comparison with a saltthat has a low salting-out effect (barium chloride).

EXAMPLE 8

Experiments were carried out with gammaglobulin as the macromolecule toshow the effect of salt contention protein sieving coefficient,diafiltration volume and yield. The solution containing gammaglobulinwas a commercial product obtained from blood plasma, Gammonativ®,supplied by Pharmacia AB, Stockholm, Sweden. Prior to filtration, thegammaglobulin solution had been purified by an intitial Cohnfractionation followed by a chromatographic stage.

The experimental conditions applied were the same as those applied inExample 1, with the exception that the virus-removing filter was aViresolve™/180 filter, the pH of the solution was 6.8 and the proteinconcentration was 2.5-5.0 A₂₈₀ units. The buffer comprised 2.2%albumin+0.15 M NaCl+0.02 M NaAc+0.075 M glycine. Total salt content:0.17 M. TABLE 12 Determining the protein sieving coefficient. ExperimentFiltrate flowrate, ml/min P/R, % 1 3.5 32 2 6.9 35 3 10.7 41 4 14.1 51 517.6 59 6 20.8 63 7 24.3 69

Determination of the protein sieving coefficient gave an optimalfiltrate flowrate of 20.8 ml/min.

EXAMPLE 9

The same conditions were applied as those applied in Example 8, with theexception that in this case the buffer comprised 2.2% albumin+1.0 MNaCl+0.02 M NaAc+0.075 M glycine. Total salt content: about 1.0 M. TABLE13 Determining the protein sieving coefficient. Experiment Filtrateflowrate, ml/min P/R, % 1 3.5 38 2 6.9 57 3 10.7 64 4 14.1 71 5 17.6 756 20.8 80 7 24.3 81

Determination of the protein sieving coefficient gave an optimalfiltrate flowrate of 20.8 ml/min.

Optimization of the yield at a filtrate flowrate of 20.8 ml/min. and aresidence time of up to 10 min gave a P/R quotient of between 60% and68%.

Diafiltration with a dilution degree of about 1 volume unit per volumeunit of entering protein solution (1+1) resulted in a yield of 90%.

EXAMPLE 10

The same conditions were applied as those applied in Example 8, with theexception that in this case the pH of the solution was 5.5. TABLE 14Determining the protein sieving coefficient. Experiment Filtrateflowrate, ml/min P/R, % 1 3.5 41 2 6.9 47 3 14.1 62 4 20.8 72 5 24.3 74

EXAMPLE 11

The same conditions were applied as those applied in Example 10, withthe exception that in this case the buffer comprised 2.2% albumin+1.0 MNaCl+0.02 M NaAc+0.075 M glycine. Total salt content: about 1.0 M. TABLE15 Determining the protein sieving coefficient. Experiment Filtrateflowrate, ml/min P/R, % 1 3.5 57 2 6.9 67 3 14.1 78 4 20.8 88 5 28.1 90

EXAMPLE 12

Experiments were carried out with albumin as the macromolecule to showthe effect of salt content on protein sieving coefficient, diafiltrationvolume and yield. The 4% solution containing Human Serum Albumin (HSA)obtained from blood plasma was supplied by Pharmacia AB, Stockholm,Sweden. Prior to filtration, the albumin-containing solution had beenpurified by combined Cohn fractionation and a chromatographic stage.

The experimental conditions applied were the same as those applied inExample 1, with the exception that the protein concentration was about10 A₂₈₀ units. The buffer comprised 0.15 M NaCl+0.02 M NaAc, resultingin a total salt content of 0.17 M. TABLE 16 Determining the proteinsieving coefficient. Experiment Filtrate flowrate, ml/min P/R, % 1 3.534 2 6.9 39 3 14.1 50 4 20.8 51 5 24.3 50

Determination of the protein sieving coefficient resulted in an optimalfiltrate flowrate of 20.8 ml/min.

EXAMPLE 13

The same conditions were applied as those applied in Example 12, withthe exception that in this case the buffer comprised 1.0 M NaCl+0.02 MNaAc, resulting in a total salt content of about 1.0 M. TABLE 17Determining the protein sieving coefficient. Experiment Filtrateflowrate, ml/min P/R, % 1 3.5 39 2 6.9 57 3 14.1 62 4 20.8 64 5 24.3 60

Diafiltration with a dilution degree of about 1 volume unit per volumeunit of entering protein solution (1+1) resulted in a yield of 85%.

EXAMPLE 14

Experiments were carried out with factor IX as the macromolecule, toshow the effect of the retention flowrate on the protein sievingcoefficient with other conditions constant. The commercial Nanotiv®solution used was similar to the solution used in Example 1. Theconditions applied were the same as those applied in Example 1, with theexception that in this case the buffer comprised 1 M NaCl+6.4 mM sodiumcitrate with a pH of 7.0. TABLE 18 Determining the protein sievingcoefficient at different retention flowrates. Retention FiltrateExperiment flowrate, l/h flowrate, ml/min P/R % 1 1 14 79 2 1 19 85 3 124 85 4 10 14 72 5 10 19 76 6 10 24 76 7 20 14 62 8 20 19 70 9 20 24 7610 30 14 65 11 30 19 69 12 30 24 73 13 40 14 60 14 40 19 64 15 40 24 7016 50 14 57 17 50 19 61 18 50 24 68 19 60 14 51 20 60 19 56 21 60 24 6222 90 14 46 23 90 19 56 24 90 24 56

Lower retention flowrates result in higher protein permeability throughthe filter.

EXAMPLE 15

Experiments were carried out with factor IX as the macromolecule in asolution having a high salt content, to show the effect of type ofvirus-filtering technique on dilution, yield, protein sievingcoefficient and process time, with other experimental conditions beingessentially constant. The experimental conditions applied, including theNanotiv® solution were the same as those applied in Example 1, with theexception of the following differences: Virus filtration techniqueTangential “Dead-end” Amount of protein solution 294 1124 prior to virusfiltration (g): Protein conc. (A₂₈₀ units): 0.66 1.0 Retention flowrate(l/h): 40 0 Filtrate flowrate buffer 24 28 (ml/min):

TABLE 19 Determining dilution, yield, protein sieving coefficient andprocess time using different virus-filtering techniques. Virusfiltration technique Tangential “Dead-end” Amount of protein solution459 1146 after virus filtration (g): Dilution: 1 + 0.56 1 + 0.02 Yield(%): 89 94 Protein sieving coefficient 17-64 92-98 (P/R in %): Actualfiltrate flowrate 15-24 7-25 (ml/min): Process time (kIU factor IX/h):31 105 Protein load 413 2360 (A₂₈₀ units/ft²):

Virus filtration of factor IX using the “dead-end” technique means lessdilution, shorter process times and results in a higher yield andprotein permeability.

EXAMPLE 16

Experiments were carried out with factor IX as the macromolecule, toshow the effect of salt content on yield and the protein sievingcoefficient when virus-filtering in accordance with the “dead-end”technique, with remaining experimental conditions constant. In additionto NaCl, the buffer also contains 6.4 mM sodium citrate (pH 7.0) in bothcases. The conditions applied, including the Nanotiv® solution were thesame as those applied in Example 1, with the exception of the followingdifferences: Salt content (M NaCl): 1.0 0.15 Amount of protein solutionprior 293 256 to virus filtration (g): Protein conc. (A₂₈₀ units): 0.840.84 Retention flowrate (l/h): 0 0 Filtrate flowrate buffer 28 28(ml/min):

TABLE 20 Determining dilution, yield and protein sieving coefficientwhen using a buffer which contained 1.0 M NaCl + 6.4 mM sodium citrate(pH 7.0). Amount of filtrate, g P/R, % Flowrate, ml/min  50 83 31 100 8228 150 84 30 200 81 23 250 81 21 Protein conc., Sample Amount, g A₂₈₀units Yield, % Prior to virus 293 0.84 100 filtration Filtrate 284 0.67 77 Wash  30 0.47  6

A total yield of 83% was obtained over the virus filter, with a dilutiondegree of 1+0.07. Process time 264 kIU factor IX/h. TABLE 21 Determiningdilution, yield, protein sieving coefficient and process time when usinga buffer containing 0.15 M NaCl + 6.4 mM sodium citrate (pH 7.0). Amountof filtrate, g P/R, % Flowrate, ml/min  50 61 22 100 62 20 150 63 18 20063 16 Protein conc., Sample Amount, g A₂₈₀ units Yield, % Prior to virus256 0.84 100 filtration Filtrate 243 0.50  56 Wash  30 0.50  7

A total yield of 63% was obtained with the virus filter, with a dilutiondegree of 1+0.07. Process time 194 kIU factor IX/h.

EXAMPLE 17

Experiments were carried out with antithrombin (AT III) as themacromolecule in a solution of low salt content, to show the effect ofthis type of virus filtration technique on dilution, yield, proteinsieving coefficient and process time, with other conditions beingessentially constant. The commercial ATenativ® solution used wasdelivered by Pharmacia AB, Stockholm, Sweden. The buffer contained 0.12M NaCl+1 mM sodium phosphate (pH 7.4) in both cases. The conditionsapplied were the same as those applied in Example 1, with the exceptionof the following differences: Virus filtration technique Tangential“Dead-end” Amount of protein solution 967 970 prior to virus filtration(g): Protein conc. (A₂₈₀ units): 9.1 9.1 Retention flowrate (l/h): 40 0Filtrate flowrate buffer 24 24 (ml/min):

TABLE 22 Determining dilution, yield, protein sieving coefficient andprocess time with the aid of different virus filtration techniques.Virus filtration technique Tangential “Dead-end” Amount of proteinsolution 1692 989 after virus filtration (g): Dilution: 1 + 0.75 1 +0.02 Yield (%): 97 97 Protein sieving coefficient 73-86 95-98 (P/R in%): Actual filtrate flowrate 15-24 8-14 (ml/min): Process time (kIU ATIII/h): 37 53 Protein load 18477 18481 (A₂₈₀ units/ft²): Filtrationefficiency 9 12 (1/m² filter * h):

Virus filtration of AT III when applying the “dead-end” technique meansless dilution, affords higher protein permeability and shorter processtimes.

EXAMPLE 18

Experiments were carried out with antithrombin (AT III) as themacromolecule, to show the effect of salt content on yield and proteinpermeability (sieving coefficient) when virus-filtering in accordancewith the tangential technique, with remaining experimental conditionsbeing constant. In addition to NaCl, the buffer contained 1 mM sodiumphosphate (pH 7.4) in all experiments. The conditions applied, includingthe ATenativ® solution, were the same as those applied in Example 17,with the exception that the retention flowrate was 20 l/h in allexperiments. TABLE 23 Determining the protein sieving coefficient atdifferent salt contents and different filtrate flowrates. Salt content,Filtrate flowrate Experiment M NaCl ml/min P/R % 1 0.15 14 79 2 0.15 1984 3 0.15 24 87 4 1.0 14 87 5 1.0 19 90 6 1.0 24 89

High salt content result in improved protein permeability with regard toAT III.

EXAMPLE 19

Experiments were carried out with Human Serum Albumin (HSA) as themacromolecule in a solution having a high salt content, to show theeffect of type of virus filtration technique on dilution, yield, proteinpermeability and process time, with other experimental conditions beingessentially constant. The HSA solution used was similar to the solutionused in Example 12. The buffer contained 1.0 M NaCl+20 mM sodium acetate(pH=7.4) in all experiments. The conditions applied were the same asthose applied in Example 1, with the exception of the followingdifferences: Virus filtration technique Tangential “Dead-end” Amount ofprotein solution 200 6460 prior to virus filtration (g): Protein conc.(A₂₈₀ units): 10 9.2 Retention flowrate (l/h): 40 0 Filtrate flowratebuffer 24 28 (ml/min):

TABLE 24 Determining dilution, yield, protein sieving coefficient andprocess time when using tangential virus filtration. Amount of filtrate,g P/R, %  50 39 100 57 200 62 300 64 350 60 Protein conc., SampleAmount, g A₂₈₀ units Yield, % Prior to virus 200 10.0 100 filtrationFiltrate 144 7.0 51 Wash 1 100 4.4 22 Wash 2 100 4.2 12

A total yield of 85% was obtained over the virus filter, with a dilutionof 1+0.72. Process time 4615 mg HSA/h. TABLE 25 Determining dilution,yield, protein sieving coefficient and process time when virus-filteringwith the “dead-end” technique. Amount of protein solution 6380 aftervirus filtration (g): Dilution: 1 + 0.0 Yield (%): 98 Protein sievingcoefficient 97-100 (P/R in %): Actual filtrate flowrate 24-34 (ml/min):Process time (mg HSA/h): 14895 Protein load 124807 (A₂₈₀ units/ft²):Filtering efficiency 34 (l/m² filter * h):

Virus filtration of HSA when applying the “dead-end” technique meansless dilution, and results in a higher yield and higher proteinpermeability and shorter process times.

EXAMPLE 20

Experiments were carried out with gammaglobulin as the macromolecule ina solution of high salt content, to show the effect of this type ofvirus-filtering technique on dilution, yield, protein sievingcoefficient and process time with remaining experimental conditionsbeing essentially constant. The gammaglobulin solution used was similarto the solution used in Example 8. The buffer contained 1.0 M NaCl+20 mMsodium acetate +0.075 M glycine (pH =5.5) in all experiments. Theconditions applied were the same as those applied in Example 1, with theexception of the following differences: Virus filtration techniqueTangential “Dead-end” Amount of protein solution 301 400 prior to virusfiltration (g): Protein conc. (A₂₈₀ units): 5.1 4.9 Retention flowrate(l/h): 40 0 Filtrate flowrate buffer 24 28 (ml/min): Transmembranepressure (bar): 0.2 0.1

TABLE 26 Determining dilution, yield, protein sieving coefficient andprocess time when using “dead-end” filtration: Amount of filtrate, gP/R, % Flowrate, ml/min 50 92 17 100 92 12 150 93 9 200 92 8 250 90 7300 88 6 350 87 5 Protein conc., Sample Amount, g A₂₈₀ units Yield, %Prior to virus 400 4.9 100 filtration Filtrate 350 4.6 82 Wash 100 2.312

A total yield of 94% was obtained over the virus filter, with a dilutiondegree of 1+0.12. Process time 2790 mg gammaglobulin/h. TABLE 27Determining dilution, yield, protein sieving coefficient and processtime when using tangential virus filtration. Amount of protein solution643 after virus filtration (g): Dilution: 1 + 1.16 Yield (%): 92 Proteinsieving coefficient 43-67 (P/R in %): Actual filtrate flowrate 16-20(ml/min): Process time (mg gammaglobulin/ 1873 h): Protein load 3192(A₂₈₀ units/ft²): Filtering efficiency 23 (l/m² filter * h):

Virus-filtration of gammaglobulin with the “dead-end” technique involvesless dilution, and results in a higher yield and protein permeabilityand shorter process times.

EXAMPLE 21

Experiments were carried out with antithrombin as the macromolecule, toillustrate that the present invention is applicable on an industrialscale by using a substatially bigger filter area (10 ft²) than in theprevious Examples (1/3 ft²). A commercial solution containingantithrombin (AT III), ATenativ®, was supplied by Pharmacia AB,Stockholm, Sweden.

Experimental Conditions:

-   Buffer: 1 M NaCl+1 mM sodium phosphate.-   Total salt content: about 1.0 M.-   Protein concentration: 9.2 A₂₈₀ units.-   Protein solution pH: 7.4.-   Amount of protein solution prior to virus filtration: 20.8 kg-   Virus separating filters: Viresolve™ /70.-   Filtering technique: dead-end.-   Filter area: 10 ft²-   Retention flowrate: 0 l/h.-   Filtrate flowrate buffer: 20 l/h

Transmembrane pressure: 0.3 bar. TABLE 28 Determining dilution, yield,protein sieving coefficient and process time when using a filter area of10 ft² and dead-end filtering technique according to the invention.Amount of protein solution 24.1 after virus filtration (kg): Dilution:1 + 0.16 Yield (%): 96 Protein sieving coefficient 94-97 (P/R in %):Actual filtrate flowrate  7-12 (ml/min): Process time (kIU AT III/h):735 Protein load 19,136 (A₂₈₀ units/ft²): Filtration efficiency 8.8(1/m² filter * h):

It is evident from this Example, that virus filtering antithrombinaccording to the invention can be applied on an industrial scale withexcellent results.

EXAMPLE 22

The virus-removing effect achieved with the experiments disclosed inExample 15 was determined by a virus study, but at a higher saltcontent. The virus-filtering technique was the “dead-end” technique. Thestudy was carried out on parvovirus, as in Example 3. The parvovirus wasadded to the solutions containing factor IX 1.0 M NaCl+0.01 M sodiumcitrate (experiment 1). The solutions were analyzed with respect to theparvovirus both before and after virus filtration. Experiment Virusreduction 1 1 × 10^(5.5)

The results show that virus filtration in accordance with Example 15using dead-end technique fulfil the requirements placed by theauthorities on the virus reduction in one process step. Furthermore, theuse of a high salt content in accordance with the invention is at leastequally as effective in removing virus as previously known techniques.

1. A filtration method comprising virus-filtering a solution containingat least one macromolecule, wherein the total salt content of thesolution is within the range of about 0.6 M up to saturation of thesolution with the salt concerned.
 2. A method according to claim 1,characterized in that the total salt content of the solution lies withinthe range of from 0.4 up to 2.5 M.
 3. A method according to claim 2,characterized in that the total salt content of the solution lies withinthe range of from 0.6 up to 2.0 M.
 4. A method according to claim 1,characterized in that the salt is selected from the group consisting ofsodium chloride, potassium chloride, sodium acetate and sodium citrateand combinations thereof.
 5. A method according to claim 1,characterized in that the macromolecule is selected from the groupconsisting of proteins, polysaccharides and polypeptides andcombinations thereof.
 6. A method according to claim 5, characterized inthat the macromolecule is factor IX.
 7. A method according to claim 5,characterized in that the macromolecule is gammaglobulin.
 8. A methodaccording to claim 5, characterized in that the macromolecule isalbumin.
 9. A method according to claim 5, characterized in that themacromolecule is antithrombin III.
 10. A method according to claim 5,characterized in that the macromolecule is a deletion derivative ofrecombinant factor VIII.
 11. A method according to claim 1,characterized in that the virus-filtering process is carried out inaccordance with the “dead-end” filtering technique.
 12. A methodaccording to claim 1, characterized in that the virus-filtering processreduces the content of non-enveloped viruses by at least 4 logs.
 13. Amethod according to claim 1, wherein a non-lipid enveloped virus isremoved from the solution.
 14. A method according to claim 13, whereinthe non-lipid enveloped virus is selected from the group consisting ofhepatitis virus A, polio virus and parvo virus.
 15. A method accordingto claim 1, wherein a lipid-enveloped virus is removed from thesolution.
 16. A method according to claim 15, wherein thelipid-enveloped virus is selected from the group consisting of hepatitisvirus B, hepatitis virus C and the human immuno-deficiency virus (HIV).17. A method according to claim 1, wherein a virus smaller than about350 nm is removed from the solution.
 18. A method according to claim 17,wherein the virus removed from the solution is smaller than 200 nm. 19.A method according to claim 18, wherein the virus removed from thesolution is smaller than 150 nm and larger than about 20 nm.